Methods and apparatus for electrophoresis of prior-cast, hydratable separation media

ABSTRACT

Methods and apparatus are presented that facilitate electrophoresis of prior-cast, hydratable separation media, usefully immobilized pH gradient (IPG) strips. The method exploits the swelling of prior-cast, hydratable separation media upon rehydration to help lodge the media in an enclosing member that permits spaced electrical communication with the enclosed separation media. The electrical communication permits a voltage gradient to be established in the enclosed separation medium sufficient to effect separation of analytes therein. Cassettes, buffer cores, electrophoresis systems and kits are presented for effecting the methods of the invention.

FIELD OF THE INVENTION

The present invention relates to methods and apparatus forelectrophoresis of prior-cast hydratable separation media. Inparticular, the invention relates to methods, cassettes, buffer cores,and systems useful for conducting isoelectric focusing using immobilizedpH gradient (IPG) strips.

BACKGROUND OF THE INVENTION

For over thirty years, isoelectric focusing (IEF) has served as aprimary tool for analyzing proteins present in complex admixture, suchas proteins present in biological samples.

In isoelectric focusing, proteins are driven by an applied electricfield through a pH gradient typically established in a support matrix,such as a gel. Proteins migrate until the isoelectric point (pI) of theprotein coincides with the local pH; at that point, the protein nolonger bears net charge and ceases to migrate, becoming focused at apoint that is characteristic of the protein.

As originally described the pH gradient for IEF was established andsustained in the gel matrix by mobile carrier ampholytes (CA). Gelstypically would be polymerized in the presence of a population of CAhaving a range of charge characteristics; upon application of a voltagegradient, the various species of CA would align themselves in the matrixto establish a pH gradient across the gel.

Although IEF with CA has proven tremendously useful, it was soondiscovered that pH gradients created by CA were susceptible to titrationby atmospheric carbon dioxide, leading to the migration of CA towardsthe cathode and destruction of the pH gradient over time, a phenomenontermed cathodic drift.

Cathodic drift can be reduced by casting IEF gels in enclosed tubes,thus limiting exposure to atmospheric CO₂. However, the tube trapsprepolymer component impurities in the matrix during polymerization,interfering with separation. Furthermore, the tube format presentsdifficulties when a second dimension of separation, such asfractionation by size, is desired.

In a different approach to the problem of cathodic drift, Bjellqvist andcolleagues immobilized the pH gradient in the support matrix, anapproach now termed immobilized pH gradient (IPG) isoelectric focusing.See Bjellqvist et al., J. Biochem. Biophys. Methods 6(4):317-39 (1982);Righetti et al., Trends Biochem. Sci. 13(9):335-8 (1988); Righetti etal., Methods Enzymol. 270:235-55 (1996); U.S. Pat. No. 4,130,470; andRighetti, Immobilized DH Gradient: Theorv and Methodology, (LaboratoryTechniques in Biochemistry and Molecular Biology, Vol. 20), ElsevierBiomedical Press, LTD, Netherlands (ASIN: 0444813012). Two-dimensionalelectrophoresis, with IPG IEF followed by size fractionation, soonfollowed. Gorg et al., Electrophoresis 9(9):531-46 (1988).

IPG not only reduced the problem of cathodic drift, but also proveduseful in reducing interference from prepolymer component impurities,since the IPG strip's plastic backing imparts sufficient structuralresilience to the gel as to permit the gel to be washed before use. Theincreased resilience also permits the gels to be stored in dehydratedform before use. Dehydrated IPG strips are today sold in a variety of pHranges and a variety of separation lengths by a number of vendors (e.g.,Immobiline DryStrip Gels, Amersham Pharmacia Biotech, Piscataway, N.J.,USA; ReadyStrip IPG, Bio-Rad Laboratories, Hercules, Calif., USA).

Problems remain, however.

Although immobilization of the gradient-forming ampholytes preventscathodic drift, the charge-bearing immobilized moieties (immobilines)remain susceptible to titration by atmospheric CO₂. CO₂ titration isexacerbated by the fact that the separation medium of IPG strips isdirectly exposed to air on at least one side. Direct exposure to airalso leads to possible dehydration of the matrix, with possible saltcrystallization, during electrophoresis.

These problems have been addressed in part by a methodologic, ratherthan structural, solution: plastic-backed IPG strips are typicallyelectrophoresed under an occlusive oil layer, which both excludes airand retards evaporation.

Use of an occlusive liquid oil layer presents its own difficulties,However. Principal among these is the requirement that electrophoresisbe performed with the IPG strip maintained in a horizontal orientation.The obligate horizontal orientation precludes use of thesmaller-footprint, vertical electrophoresis devices typically used forSDS-polyacrylamide gel electrophoresis (SDS-PAGE), such as thosedescribed in Tippins et al., U.S. Pat. No. 5,888,369. In addition, theuse of oil requires deft manual technique and proves time-intensive.

Wiktorowicz et al., U.S. Pat. No. 6,013,165, describe an apparatus inwhich immobilized pH gradient isoelectric focusing can be performedwithout use of a liquid oil layer. A continuous pKa gradient isimmobilized on at least one of the major opposing surfaces of a cavityformed between two plates. The cavity, which can be further segmentedinto parallel channels, is then filled with a flowable separationmedium. Electrophoresis is preferably conducted with the assemblyoriented horizontally to minimize convection currents in the flowableseparation medium. The apparatus does not readily permit insertion ofprior-cast hydratable separation media, such as commercial IPG strips,nor does it readily permit electrophoresis in the vertical dimension.

There thus exists a need in the art for methods and apparatus that allowIPG strips, and other prior-cast hydratable separation media, to beelectrophoresed without requiring contact with an occlusive fluid oillayer. There further exists a need in the art for methods and apparatusthat allow IPG strips, and other prior-cast hydratable separation media,to be electrophoresed in a vertical orientation.

SUMMARY OF THE INVENTION

The present invention solves these and other needs in the art byproviding methods, apparatus, and kits for electrophoresis of prior-casthydratable separation media that obviate the use of an occlusive oillayer, thereby obviating the requirement that electrophoresis beperformed in the horizontal orientation.

The present invention is based, in part, upon the discovery that theswelling that attends rehydration of prior-cast hydratable separationmedia can be exploited to help lodge such media in an enclosure thatpermits spaced electrical communication with the enclosed separationmedium. The spaced electrical communication makes it possible to apply avoltage gradient to the prior-cast hydratable separation media while themedium is otherwise enclosed, permitting electrophoresis to be conductedwithin a cassette.

Enclosed, the separation medium's contact with air is substantiallyreduced. In cases in which the prior-cast hydratable separation mediumis an IPG strip, the reduction in air contact obviates the prior artrequirement for occlusive contact with a fluid oil layer duringimmobilized pH gradient isoelectric focusing.

Enclosed, and lacking an attendant fluid oil layer, the prior-castseparation medium can be electrophoresed in any physical orientation. Incases in which the prior-cast hydratable separation medium is an IPGstrip, relaxation of the prior-art requirement for horizontalelectrophoresis makes it newly possible to perform IPG electrophoresisusing the widely distributed, small footprint, vertical electrophoresisgel boxes presently used to perform SDS-PAGE.

Thus, in a first aspect, the invention provides a method for performingelectrophoresis, comprising: hydratingly lodging a prior-cast hydratableelectrophoretic separation medium within an enclosing member thatpermits spaced electrical communication with the enclosed medium; andthen using the spaced electrical communication to establish a voltagegradient in the enclosed separation medium sufficient to effectelectrophoretic separation of analytes therein.

In one embodiment, the method further comprises the antecedent step ofinserting the prior-cast hydratable electrophoretic separation medium inits dehydrated state into the enclosing member. In another embodiment,the method further includes a later step of removing the prior-casthydratable electrophoretic separation medium from the enclosing member.The medium once removed can be used, for example, to apply theone-dimensionally fractionated sample to a gel to effect a seconddimension of separation.

In some embodiments, the step of hydratingly lodging comprises:contacting the dehydrated prior-cast hydratable electrophoreticseparation medium with an aqueous solution, often an aqueous solutionthat includes the sample to be fractionated.

The methods of the present invention are particularly useful inperforming isoelectric focusing using immobilized pH gradient strips.Thus, the prior-cast hydratable electrophoretic separation medium usedin the practice of the present invention can usefully have animmobilized pH gradient.

As described above, the methods of the present invention include the useof an enclosing member that has (i) means for hydratingly lodging aprior-cast electrophoretic separation medium therewithin, and (ii) meansfor spaced electrical communication with the enclosed separation medium,wherein the spaced electrical communication means can be used to apply avoltage gradient to the enclosed medium sufficient to effectelectrophoretic separation of analytes present therewithin.

Thus, in another aspect, the invention provides a cassette forperforming electrophoresis, comprising: means for hydratingly lodging aprior-cast electrophoretic separation medium within an enclosing member;and means for spaced electrical communication with the enclosed medium,wherein the spaced electrical communication means can be used toestablish a voltage gradient in the separation medium sufficient toeffect electrophoretic separation of analytes therein.

In certain embodiments, the cassette of the present invention comprises:a form-retaining member, and at least one channel, wherein theform-retaining member imparts dimensional integrity to the channel orchannel(s). In typical embodiments, the cassette includes a plurality ofsuch channels.

Each channel present in the cassette and useful for performing themethods of the present invention has a first channel entry, a secondchannel entry, and a cavity therebetween, the channel cavity being sodimensioned as to permit insertion of a hydratable prior-castelectrophoretic separation medium in its dehydrated state and lodginglyenclose the strip in its rehydrated state. The first and second channelentries permit spaced electrical communication with the channel cavity;the spaced electrical communication permits current to be flowed throughthe channel cavity.

In some embodiments, the form-retaining member contributes the entirecircumferential wall of the cavities of the channels. In other,multilaminate embodiments, the cassette further comprises a laminatecover; the laminate cover adheres directly or indirectly to theform-retaining member and contributes at least part of thecircumferential wall of said channels. In these latter embodiments, theadherence of the laminate cover to the form-retaining member istypically reversible.

In other embodiments, the cassette further comprises a firstwell-forming member, which adheres directly or indirectly to theform-retaining member, and which defines fluid reservoirs at a pluralityof first channel entries. Usefully, the cassette can further comprise asecond well-forming member, the second well-forming member adhering,directly or indirectly, to the form-retaining member and defining fluidreservoirs at a plurality of second channel entries. When present, thewell-forming members can usefully be reversibly adherent to theform-retaining member.

In one series of related embodiments, the first and second channelentries for each of the channels permit electrical communication withthe intervening channel cavity through a common surface of the cassette.In another series of related embodiments, the first and second channelentries permit electrical communication with their intervening cavitythrough separate surfaces of the cassette. These two mutually exclusivegeometries call for different electrode geometries, and thus differentelectrophoresis buffer cores, to complete the circuits required forelectrophoresis.

The prior-cast hydratable electrophoretic separation medium can beprovided by the user, can be included within one or more channels of thecassette without requirement for user insertion thereof, or can beprovided separately packaged with the cassette in a kit.

As to the latter, it is another aspect of the present invention toprovide kits for facilitating electrophoresis of prior-cast hydratableelectrophoretic separation media. The kits typically comprise a cassetteof the present invention and at least one prior-cast hydratableelectrophoretic separation medium suitably dimensioned as to behydratingly lodgeable in said cassette.

In some embodiments, the kit includes a cassette and at least oneconductive wick for use therewith; often, in such kits, a sufficientnumber of wicks are provided to facilitate both anodic and cathodicconnections with the cassette.

The cassettes of the present invention can be used to effect verticalelectrophoresis of prior-cast hydratable separation media, usefully inthe buffer tanks that are commonly used, with buffer cores, for SDS-PAGEelectrophoresis. In cassette embodiments in which the first and secondchannel entries open to separate surfaces of the cassette, buffer coresresently used for SDS-PAGE electrophoresis can be used. In cassetteembodiments in which the first and second channel entries open to thesame surface of the cassette, alternative buffer core geometries arerequired.

Thus, it is another aspect of the present invention to provide a buffercore for vertical electrophoresis of pre-cast hydratable electrophoreticseparation media, comprising: a substantially inflexible frame, ananode, and a cathode in spaced relationship to the anode. The buffercore frame has a first cassette engagement face and a second cassetteengagement face. Operational engagement of a first and second cassetteto the respective first and second frame engagement faces creates achamber internal to the frame that is sealed on 5 sides. The cathode andanode are each in electrical communication with the interior of theinternal chamber, and operational engagement of a first and secondcassette to the respective first and second frame engagement facescauses spaced contact of the anode and cathode to the surface of atleast one cassette that engages the frame engagement surface, allowingelectrophoresis of prior-cast hydratable separation media enclosedtherein.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and advantages of the present invention willbe apparent upon consideration of the following detailed descriptiontaken in conjunction with the accompanying drawings, in which likecharacters refer to like parts throughout, and in which:

FIG. 1 is a front perspective view of a cassette of the presentinvention;

FIG. 2 is a front perspective view of a cassette of the presentinvention with an IPG strip inserted into one of six available channels;

FIG. 3A is a front Perspective view of a cassette of the presentinvention with a first conductive wick contacting the anodic end of IPGstrips present in three of six available channels and a secondconductive wick contacting the cathodic end of the three IPG strips;

FIG. 3B is a back perspective view of a cassette of the presentinvention, particularly showing a recessed region that facilitates heatdissipation during electrophoresis;

FIG. 4 is an exploded side perspective view of a multilaminate cassetteof the present invention;

FIG. 5 is an exploded side perspective view of a loading well assemblyof a cassette of the present invention;

FIG. 6A is a front perspective view of a buffer core of the presentinvention (front) operationally aligned to contact its anode and cathodeelectrodes respectively to anodic and cathodic wicks of a cassette ofthe present invention (rear);

FIG. 6B is a front perspective view of the buffer core and cassette ofFIG. 6A in operational contact with one another;

FIG. 6C shows a buffer core of the present invention, with cassettes ofthe present invention operationally engaged thereupon, further engagedin an electrophoresis chamber;

FIG. 7A is a front view of a cassette of the present invention in whichchannel entries open through opposite surfaces of the cassette;

FIG. 7B is a side view of the cassette of FIG. 7A;

FIG. 7C is an exploded perspective view of two cassettes as shown inFIGS. 7A and 7B showing their operational relationship to a prior artbuffer core;

FIG. 7D is a perspective view of the cassettes of FIGS. 7A and 7B inoperational contact with a prior art buffer core; and

FIG. 8 shows IPG strips after electrophoresis in channels of the statedinternal dimensions.

DETAILED DESCRIPTION

The present invention is based, in part, upon the discovery that theswelling that attends rehydration of prior-cast hydratable separationmedia can be exploited to help lodge such media in an enclosure thatpermits spaced electrical communication with the enclosed separationmedium. The spaced electrical communication makes it possible to apply avoltage gradient to the prior-cast hydratable separation media while themedium is lodged within the enclosing member.

Enclosed, the separation medium's contact with air is substantiallyreduced. In cases in which the prior-cast hydratable separation mediumis an IPG strip, the reduction in air contact obviates the prior artrequirement for occlusive contact with a fluid oil layer duringimmobilized pH gradient isoelectric focusing.

Enclosed, and lacking an attendant fluid oil layer, the prior-castseparation medium can be electrophoresed in any physical orientation. Incases in which the prior-cast hydratable separation medium is an IPGstrip, relaxation of the prior-art requirement for horizontalelectrophoresis makes it newly possible to perform IPG electrophoresisusing the widely distributed, small footprint, vertical electrophoresisgel boxes presently used to perform SDS-PAGE.

In a first aspect, therefore, the invention provides a method forperforming electrophoresis, particularly for performing electrophoresisusing prior-cast, hydratable separation media. As used herein, the term“electrophoresis” explicitly includes isoelectric focusing.

In a first step, the method comprises hydratingly lodging a prior-casthydratable electrophoretic separation medium within an enclosing memberthat permits spaced electrical communication with the enclosed media. Ina second step, the spaced electrical communication is used to apply avoltage gradient to the enclosed medium sufficient to effectelectrophoretic separation of analytes therein.

As used herein, the phrase “prior-cast electrophoretic separationmedium” (and equivalently, “prior-cast separation medium”) refers to anelectrophoretic separation medium, typically a polymeric gel, that hasfirst been solidified, or gelled, elsewhere than in the enclosing memberin which electrophoresis is to be performed.

Electrophoretic separation media, and methods of preparing, casting, andperforming electrophoresis using electrophoretic media, are well knownin the analytical arts, and need not be detailed here. See, e.g.,Rabilloud (ed.), Proteome Research: Two-Dimensional Gel Electrophoresisand Identification Methods, Springer Verlag, 2000 (ISBN: 3540657924);Westermeier, Electrophoresis in Practice, 2nd ed., John Wiley & Sons,2000 (ISBN 3527300708); B. D. Hames et al. (eds.), Gel Electrophoresisof Proteins, 3rd ed., Oxford University Press, 1998) (ISBN 0199636419);and Jones, Gel Electrophoresis: Nucleic Acids: Essential Techniques,(John Wiley & Son Ltd. 1996) (ISBN 0471960438), the disclosures of whichare incorporated herein by reference in their entireties.

Although polyacrylamide (that is, a polymerization product of acrylamidemonomer crosslinked with N,N′-methylenebisacrylamide) and agarose arethe two polymeric gels most commonly used in electrophoresis today, thepresent invention proves useful in electrophoresing a far wider varietyof polymeric gels.

Because the gel is first solidified, or gelled, elsewhere than in theenclosing member in which electrophoresis is to be performed, the“prior-cast electrophoretic separation medium” used in the presentinvention must have sufficient structural resiliency to be transferredor released from its casting mold and thereafter lodged within theenclosure of the present invention.

Typically, such structural resiliency will be imparted to the separationmedium by the adherence thereto or incorporation therein of a layer orlamina of another material, such as plastic. Such layers are known inthe art, and include, e.g., polyester film backings, as are found incommercial IPG strips, and polyester mesh fabric, which can beincorporated into the separation medium.

Although the “prior-cast electrophoretic separation medium” used in thepresent invention is typically fashioned as a strip—that is, with afirst dimension substantially greater than a second dimension—suchdimensions are not required for practice of the present invention.Nonetheless, for ease of description, all prior-cast electrophoreticseparation media useful in the practice of the present invention arereferred to in the alternative herein as “strips”.

A “prior-cast hydratable electrophoretic separation medium” is aprior-cast electrophoretic medium that can be dehydrated and that, afterrehydration, has retained sufficient structural integrity to permitelectrophoretic separation of analytes there within.

Neither complete removal of moisture, during dehydration, nor completesaturation with liquid, during rehydration, is required or intended. Itsuffices for practice of the present invention that the prior-cast,hydratable, electrophoretic separation medium swell detectably aftercontact in its dehydrated state with an aqueous solution (“aqueousbuffer”, “buffer”).

Typically, the prior-cast hydratable electrophoretic separation mediumwill swell at least about 5% in volume, often at least about 10%, 15%,20%, even at least about 25%, 30%, 40% or more in volume upon contactwith an aqueous buffer. The volume increase can be manifest in all threedimensions or, when the separation medium is backed with an inextensiblelayer, principally in one or in two dimensions. The volume increase canoccur over a period of minutes or, in the case of IPG strips, moretypically over a period of hours.

The degree of swelling is sufficient if the prior-cast, hydratable,electrophoretic separation medium swells sufficiently upon contact withan aqueous solution (“aqueous buffer”, “buffer”) as to permit hydratablelodging in an enclosing member.

By “hydratable lodging” is intended that the prior-cast, hydratableseparation medium be insertable into an enclosing member in itsdehydrated state, and that it become lodged in the enclosing member inits rehydrated state.

Although the strip must be “insertable” in its dehydrated state, thestrip need not necessarily be removable from the enclosing member in itsdehydrated state.

The rehydrated prior-cast hydratable separation medium is said to be“lodged” in the enclosing member (equivalently, “lodgingly enclosed”therein) when two conditions are met. First, the strip remains withinthe enclosing member when the enclosing member is brought into verticalorientation. Second, when the enclosing member is brought into verticalorientation, at least 50% of the separation medium is precluded fromdirect communication with ambient atmosphere. Furthermore, althoughfrictional and surface tension forces between the rehydrated separationmedium and the enclosing member can contribute to the strip's lodgingtherein, it is not intended that such frictional or surface tensionforces be sufficient in themselves to effect lodging of the strip withinthe enclosing member.

The enclosing member will be sufficiently form-retaining as to be ableto maintain dimensional integrity when maintained in contact with aprior-cast, hydratable separation medium that is swelling. In certainembodiments described in detail below, the enclosing member is acassette having a form-retaining channel cavity within which theprior-cast, hydratable separation medium is engaged.

The enclosing member further permits spaced electrical communicationwith the enclosed prior-cast hydratable separation medium. Communicationcan be direct, as by through-passage of anode and cathode electrodes, orindirect, as by passage of current through an intermediate polymer layeror wick, as will be further discussed below.

After the prior-cast hydratable electrophoretic separation medium islodged in the enclosing member, the spaced electrical communication isused to apply a voltage gradient sufficient to effect electrophoreticseparation of analytes therein.

Although described particularly herein as application of a voltagegradient to the separation medium, it is understood that current isthereby caused to flow through the separation medium, and that themethod could equally be described as flowing current through theseparation medium.

The electrical parameters to be used depend upon the composition anddimensions of the enclosed electrophoretic medium, the composition ofthe sample, the composition of the rehydration solution, and the type ofdesired separation, and is thus determined using factors well known inthe electrophoretic arts.

For example, in cases in which the prior-cast hydratable electrophoreticmedium is a 70 mm Immobiline DryStrip gel having pH range of 4-7(Amersham Pharmacia Biotech, Piscataway, N.J., USA), a typical protocolwould be to apply 200 V for 1 minute, ramping up to 3500 V over 1½hours, followed by 3500 V for 55 minutes to 1½ hours, all with currentlimited to 2 mA. Other protocols can be found, e.g., in 2-DElectrophoresis Using Immobilized pH Gradients: Principles and Methods,Amersham Pharmacia Biotech (part 80-6429-60; Rev. A, September 1998),the disclosure of which is incorporated wherein by reference in itsentirety.

Returning to the method in more detail, the prior-cast hydratableelectrophoretic separation medium typically inserted by the user in itsdehydrated state in the enclosing member.

By way of example, in embodiments further described below, theprior-cast hydratable separation medium, such as an IPG strip, ismovably inserted by hand into a channel cav ity present within theenclosing member. As another example, where the enclosing member ishinged, or otherwise reversibly separable, the prior-cast hydratableseparation medium, such as an IPG strip, is movably inserted by handinto a depression, with the channel cavity thereafter completed byclosing the member.

Although typical, movable insertion of the dehydrated strip into theenclosing member is not always required. For example, the dehydratedstrip can be earlier-inserted during manufacture of the enclosingmember, obviating insertion of the dehydrated prior-cast separationmedium into the enclosing member by the user.

The dehydrated separation medium is then contacted with an aqueoussolution.

The composition of the rehydration solution will depend upon thecomposition of the sample and separation medium and the intendedelectrophoretic procedure, and its choice will thus depend on factorsthat are well known in the electrophoretic arts.

For example, where the prior-cast hydratable separation medium is acommercial IPG strip, such as an Immobiline DryStrip (Amersham PharmaciaBiotech, Piscataway, N.J., USA), the rehydration solution can usefullyinclude urea, non-ionic or zwitterionic detergents, dithiothreitol(DTT), dye, and a carrier ampholyte mixture suited to the pH range ofthe IPG strip. Carrier ampholyte mixtures for use in such rehydrationsolutions are available commercially (e.g., IPG Buffer pH 3.5-5.0, cat.no. 17-6002-O₂; IPG Buffer pH 4.5-5.5, cat. no. 17-6002-04; IPG BufferpH 5.0-6.0, cat. no. 17-6002-05; IPG Buffer pH 5.5-6.7, cat. no.17-6002-06; IPG Buffer pH 4-7, cat. no. 17-6002-86; IPG Buffer pH 6-11,cat. no. 17-6002-78; IPG Buffer pH 3-10 NL, cat. no. 17-6002-88; IPGBuffer pH 3-10, cat. no. 17-6002-87, all from Amersham PharmaciaBiotech, Piscataway, N.J., USA).

The rehydration solution can also advantageously include the sampleintended to be separated in the prior-cast hydratable separation medium.

For example, in cases in which the prior-cast hydratable electrophoreticseparation medium is an IPG strip, the sample to be separated can be amixture of proteins, such as those from a biological sample, and canusefully be or have been denatured, as by chaotropes, reducing agents,and detergents. In cases in which the separation medium is other than animmobilized pH gradient strip, the sample can include other types ofmacromolecules, such as nucleic acids.

The methods of the present invention can include the later step ofremoving the prior-cast hydratable separation medium from the enclosingmember after electrophoresis. The method of removal will depend on thestructure of the enclosing member, as will be further described below.As an alternative to removal, the separation medium in certainembodiments of the methods of the present invention can be furtheranalyzed within the enclosing member, such as by staining and drying.

As described above, the methods of the present invention include the useof an enclosing member that has (i) means for hydratingly lodging aprior-cast electrophoretic separation medium therewithin and (ii) meansfor spaced electrical communication with the enclosed separation medium,wherein the spaced electrical communication means can be used to apply avoltage gradient to the enclosed separation medium sufficient to effectelectrophoretic separation of analytes present therewithin.

It is, therefore, another aspect of the present invention to provide anenclosing member useful in the practice of the methods of the presentinvention, which enclosing member is hereinafter called a “cassette”.

FIG. 1 is a front perspective view of an embodiment of a cassette of thepresent invention.

Cassette 100 comprises form-retaining member 10 and at least one channel12 (in the embodiment shown in FIG. 1, cassette 100 has sixsubstantially parallel channels 12, although fewer or greater numberscan be present). Form-retaining member 10 imparts dimensional integrityto prior-formed channels 12.

Referring again to FIG. 1, channel 12 has first channel entry 14 andsecond channel entry 16 and cavity 18 therebetween. Cavity 18 of channel12 is so dimensioned as to movingly engage a prior-cast hydratableelectrophoresis medium (“strip”), such as an IPG strip, in itsdehydrated state, and to lodgingly enclose the strip after hydrationthereof.

First channel entry 14 and second channel entry 16 permit electricalcommunication with cavity 18, and thus define a channel current flowaxis through cavity 18. In certain embodiments of cassette 100particularly designed for use with buffer cores of the prior art (seebelow), the channel current flow axis is in a plane substantiallyparallel to a substantially planar first surface of form-retainingmember 10.

To use cassette 100 in the methods of the present invention,rehydratable electrophoresis strip 20, such as an IPG strip, is insertedin its dehydrated state into channel 12, typically through entry 14 orentry 16. In alternative embodiments, strip 20 has been prior-insertedinto cassette 100, either by the user or by the manufacturer thereof.

Strip 20 is rehydrated within channel 12 by application of a rehydrationsolution, optionally containing the sample to be fractionated.

Rehydration solution is typically dispensed into channel 12 prior toinsertion of strip 20, since insertion of strip 20 into channel 12 isfacilitated by wetting of the interior of channel 12. Strip 20 can,however, be prior-inserted into channel 12, with rehydration solutionthereafter applied at either or both of entries 14 and 16. For samplesrequiring long rehydration times, entry 14, entry 16, or both can besealed—e.g. with tape or cover slip—to prevent evaporation and theaccidental discharge of rehydration solution.

Upon rehydration, strip 20 becomes lodged in cavity 18 of channel 12, atleast in part due to swelling of the separation medium. Strip 20 isthereafter not readily removed from channel 12 without expansion ofcavity 18, as further described below.

If the sample to be electrophoretically fractionated is not included inthe rehydration solution, sample is then applied at entry 14, entry 16,or both with the cassette oriented horizontally to retain sample, andallowed to enter the separation medium. Alternatively, sample can beprior-absorbed into a wick which is then inserted into entry 14, entry16, or both, from which wick the sample then enters the separationmedium. As further described below, sample entry can be facilitated byapplication of electrical current.

Electrophoresis is then performed by applying a voltage gradient tostrip 20, causing current to flow along the channel current flow axis.

Thereafter, strip 20 is typically removed from channel 12 for furtherprocessing, such as staining and/or contacting of strip 20 (or a portionthereof) to a gel to effect separation along a second dimension. Removalis typically effected by expansion of cavity 18 using a methodappropriate to the composition of cassette 100; for example, inembodiments of cassette 100 in which one or more laminae contribute tothe circumferential walls of cavity 18, removal can be effected bypeeling of the laminae, thus opening channel 12. For certain purposes,further processing can be effected within channel 12.

Returning to FIG. 1, form-retaining member 10 is constructed ofform-retaining nonliquid materials. Preferred materials are those thatare readily machined, molded, or etched, that are chemicallycompatible—that is, do not suffer substantial degradation uponcontact—with electrophoretic buffer systems, that do not appreciablybind or impede the transport of analytes through the enclosed gel, andthat provide a vapor gas barrier. Usefully, form-retaining member 10 canbe constructed from translucent, or transparent material, includingoptical quality transparent material, thus permitting strip 20 to bevisualized while engaged in cavity 18. Typically, form-retaining member10 is constructed of materials that are substantially electricallynonconducting, thus reducing or eliminating the concurrent action onstrip 20 of electrical fields other than those along the channel currentflow axis through cavity 18.

In typical embodiments, form-retaining member 10 is composed of ceramic,quartz, glass, silicon and its derivatives, plastic, or mixturesthereof. Among plastics useful in the construction of form-retainingmember 10 are polymethylacrylic, polyethylene, polypropylene,polyacrylate, polymethylmethacrylate, polyvinylchloride,polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal,polysulfone, celluloseacetate, cellulosenitrate, nitrocellulose,polystyrene, polyacrylonitrile, polyurethane, polyamides, polyaniline,polyester, and mixtures or copolymers thereof.

Form-retaining member 10 is also usefully composed of materials thatpermit heat to be conducted away from strip 20 during electrophoresis.In that regard, form-retaining member 10 can usefully be shaped toinclude recessed region 27, shown in FIG. 3A and particularly in FIG.3B, reducing the thickness of form-retaining member 10 in regionsproximal to channels 12, reducing-thermal resistance between strip 20and a heat sink, usefully a fluid filled chamber, as further discussedbelow.

Form-retaining member 10 confers dimensional integrity upon channels 12.Dimensional integrity is important to permit the dispensing into channel12 of rehydration solution (optionally with sample to be fractionated),to permit strip 20 to be inserted into channel 20, and to effecthydratable lodging of strip 20 in channel 12 upon rehydration.

Form-retaining member 10 can confer dimensional integrity upon channel12 by contributing at least a portion of the circumferential wall ofcavity 18 of channel 12.

For example, cavity 18 of channel 12 can be constructed as a tunnel,bore, or conduit within form-retaining member 10. In such embodiments,form-retaining member 10 contributes the entirety of the circumferentialwall of cavity 18.

Alternatively, cavity 18 can be partially enclosed within form-retainingmember 10, with only a portion of the circumferential cavity wall ofcavity 18 contributed by member 10. In these latter embodiments,channels 12 can be machined into form-retaining member 10, or, dependingon the composition of form-retaining member 10, lithographed, engraved,isotropically or anisotropically etched, milled, mechanically orchemically polished, or molded into form-retaining member 10.Alternatively, in these latter embodiments channels 12 can be fabricatedon form-retaining member 10 from silicon or resin deposits or slabs.

In embodiments in which cavities 18 are not fully enclosed by inflexiblemember 10, channels 12 can be rendered fluidly enclosing along cavity 18by physical attachment to form-retaining member 10 of one or moreadditional laminae.

FIG. 4 is an exploded side perspective view of a multilaminateembodiment of cassette 100 of the present invention.

In the embodiment shown in FIG. 4, form-retaining member 10 includesdepression 13. Laminate cover 42 includes a plurality of entries 50.Upon attachment of laminate cover 42 to form-retaining member 10,depression 13 becomes fluidly enclosing along cavity 18, thus completingchannel 12, with entries 50 contributing to channel entries 14 and 16.

As with form-retaining member 10, laminate cover 42 can usefully beoptically translucent or transparent, and is usefully substantiallyelectrically insulating.

As with form-retaining member 10, laminate cover 42 can be composed ofceramic, quartz, glass, silicon and its derivatives, alumina, polymer,plastic, or mixtures thereof. Among plastics useful in the constructionof laminate cover 42 are polymethylacrylic, polyethylene, polypropylene,polyacrylate, polymethylmethacrylate, polyvinylchloride,polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal,polysulfone, celluloseacetate, cellulosenitrate, nitrocellulose,polystyrene, polyacrylonitrile, polyurethane, polyamides, polyaniline,polyester, and mixtures and copolymers thereof.

Laminate cover 42 can usefully be, and is often preferably, flexible.Although laminate cover 42 can be of any thickness, to conferflexibility laminate cover 42 can usefully be a film.

Laminate cover 42 can be attached to form-retaining member 10 by bondingmeans known in the microfabrication arts, including thermal welding,ultrasonic welding, and application of adhesives or adhesive layers.

For example, U.S. Pat. Nos. 5,800,690 and 5,699,157, incorporated hereinby reference in their entireties, describe methods for completingchannels by attaching planar cover elements to micromachined substratesby thermal bonding, application of adhesives, or by natural adhesionbetween the two components. U.S. Pat. No. 5,593,838, incorporated hereinby reference, teaches that localized application of electric fieldspermits the meltable attachment of a cover element at about 700° C.,well below the flow temperature of silicon (about 1400° C.) or ofCorning 7059 glass (about 844° C.). WO 96/04547 (Lockheed Martin EnergySystems), incorporated herein by reference in its entirety, teaches thata cover plate can be bonded directly to a glass substrate aftertreatment in dilute NH₄OH/H₂O₂, followed by annealing at 500° C., wellbelow the flow temperature of silicon-based substrates. WO 98/45693(Aclara Biosciences), incorporated herein by reference in its entirety,discloses a thermal bonding method for fabricating enclosed microchannelstructures in polymeric, particularly plastic, substrates, an adhesivemethod in which adhesive is applied in a film no more than 2 μm thick,and methods in which fluid curable adhesives are rendered nonflowable bypartial curing before apposition of adherends.

Laminate cover 42 is usefully attached to form-retaining member 10 byreversible bonding means, thus permitting the user to separate laminatecover 42 from form-retaining member 10 after completion ofelectrophoresis, which in turn permits strip 20 to be removed fromchannel 12 for further processing. Constructing laminate cover 42 as aflexible film offers advantages in such user-mediated separation oflaminate cover 42 from form-retaining member 10.

In the embodiment depicted in FIG. 4, laminate cover 42 is attachedadhesively to form-retaining member 10 using double-sided laminateadhesive layer 46.

As shown, double-sided laminate adhesive layer 46 has elongate slots 48that are congruent with depressions 13. Such slots 48 prevent contactbetween double-sided adhesive layer 46 and strip 20 when strip 20 ismovably inserted into channel 12; contact with adhesive can interferewith movable insertion of strip 20 into cassette 100.

In multilaminate embodiments of cassette 100 in which laminate cover 42is attached with a double-sided adhesive layer 46, the thickness ofadhesive layer 46 can be adjusted to change the internal diameter ofcavity 18 of channel 12, thus accommodating hydratable strip media ofdifferent thicknesses.

In alternative multilaminate embodiments of cassette 100, laminate cover42 is itself fashioned as a form-retaining member, typically thickerthan the flexible film above-described. In some of these embodiments,laminate cover 42 is fashioned as a discrete structure. In otherembodiments, form-retaining laminate cover 42 and form-retaining member10 are movably attached to one other, as by a hinge, or plurality ofhinges, present therebetween. The hinge need not itself be fashioned asa separate, intermediating, structure, but can instead be fashioned as afoldable seam between form-retaining member 10 and laminate cover 42.Such seams are common in plastic cases designed to hold, e.g., drillbits.

In cases in which laminate cover 42 is form-retaining, it can beassembled to form-retaining member 10 by, e.g., snapping laminate cover42 to form-retaining member 10. A pressure compliant surface, onform-retaining member 10 and/or laminate cover 42, facilitates sealingof the two layers, forming an enclosing member suitable forelectrophoresis. Although assembly by snapping of laminate cover 42 toform-retaining member 10 has been described with particularity, anyother mechanical engagement approach, such as mating of tongue andgroove, insertion of a tab into a slot, etc., can also be used tosimilar effect.

In multilaminate embodiments of cassette 100—both those with flexibleand those with form-retaining laminate covers—the internal diameter ofcavities 18 can be adjusted by adjusting the depth of incursion ofchannel 12 into form-retaining member 10. In multilaminate embodimentsof cassette 100 in which laminate cover 42 is thicker than a film, theinternal diameter of cavities 18 can be adjusted additionally byadjusting the depth of incursion of channel 12 into laminate cover 42.

Channel 12 is so dimensioned—in both multilaminate and unitaryembodiments of cassette 100—as to permit insertion of a prior-casthydratable strip-based electrophoresis medium, such as an IPG strip, inits dehydrated state, and to lodgingly enclose the strip afterhydration.

Immobiline DryStrip IPG strips, presently available commercially fromAmersham Pharmacia Biotech, (Piscataway, N.J., USA), have an approximatewidth of 3 mm and a depth of 0.5 mm. Accordingly, to permitelectrophoresis of these commercial IPG strips, channel 12 of cassette100 will have a width of at least about 3.0 mm, 3.1 mm, 3.2 mm, 3.3 mm,3.4 mm, and even 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4.0 mm, andeven 4.1 mm, and will have depth of at least about 0.5 mm, 0.6 mm, 0.61mm, 0.62 mm, 0.63 mm, 0.64 mm, 0.65 mm, 0.66 mm, 0.67 mm, 0.68 mm, 0.69mm, and even 0.7 mm, 0.71 mm, 0.72 mm, 0.73 mm, 0.74 mm, 0.75 mm, 0.76mm, and even 0.77 mm so as to movingly engage such strips in theirdehydrated state and lodgingly enclose the strips when rehydrated.

ReadyStrip IPG strips, presently available commercially from Bio-Rad(Hercules, Calif., USA) have strip width of 3.3 mm and gel thickness of0.5 mm. Accordingly, to permit electrophoresis of these commercial IPGstrips, channel 12 of cassette 100 will have an approximate width of atleast about 3.3 mm, 3.4 mm, and even and even 3.5 mm, 3.6 mm, 3.7 mm,3.8 mm, 3.9 mm, 4.0 mm, and even 4.1 mm, and will have depth of at leastabout 0.5 mm, 0.6 mm, 0.61 mm, 0.62 mm, 0.63 mm, 0.64 mm, 0.65 mm, 0.66mm, 0.67 mm, 0.68 mm, 0.69 mm, and even 0.7 mm, so as to movingly engagesuch strips in their dehydrated state and lodgingly enclose the stripswhen rehydrated.

In a presently preferred embodiment, suitable for electrophoresis ofstrips from both manufacturers, channels 12 have width of 3.7 mm anddepth of 0.64 mm.

As would be expected, prior-cast hydratable electrophoretic separationmedia can, and likely will, be manufactured with dimensions differentfrom those presently used. Accordingly, cassettes 100 of the presentinvention are not limited to those dimensioned for use with theabove-described strips.

Design of the internal dimensions of channel 12, so as to permitinsertion of prior-cast hydratable strip based media in their dehydratedstate and lodgingly enclose the strips when rehydrated, is well withinthe skill in the art.

A simple test for suitability of the internal dimensions of channel 12for a prior-cast hydratable electrophoretic separation medium 20 of anygiven depth and width is as follows:

-   -   (1) Position the cassette horizontally and fill channel 12 with        water;    -   (2) Insert strip 20 through entry 14 or through entry 16 into        channel 12 and advance as far as possible by hand;    -   (3) After 8 hours, bring cassette 100 to the vertical position        with the visibly labeled entry superior, and observe.        Dimensions of channel 12 are suitable if, in step (2), strip 20        can be advanced into channel 12 to a point at which less than 1        cm of strip 20 remains outside the entry chosen for insertion,        and if, in step (3), air does not directly contact more than 50%        of the enclosed separation medium.

At one end of the useable spectrum of channel dimensions, the swellingof the separation medium causes direct, occlusive, contact of theseparation medium with the channel's internal wall along substantiallyall of the channel cavity. In this case, a visibly labeled solution(such as 0.2% w/v bromphenol blue in water) applied to the superiorchannel entry will be substantially precluded from the channel cavity.That is, a visibly labeled solution will typically not extend more thanabout 0.25 cm beyond the channel entry into the channel cavity. At theother end of the useable spectrum of channel dimensions, the swelling ofthe separation medium is insufficient to cause occlusive contact of theseparation medium with the channel's internal wall along substantiallyall of the channel cavity. In this latter case, a visibly labeledsolution such as 0.2% w/v bromphenol blue in water will enter thechannel cavity from the superior entry when the cassette is broughtvertical. In neither case, however, will air contact more than 50% ofthe enclosed separation medium.

An additional, functional test for suitability of the internaldimensions of channel 12 for a prior-cast hydratable electrophoreticseparation medium of given dimensions is to replace step (3) of the testset forth above with an actual electrophoresis experiment; dimensions ofchannel 12 are suitable if, in step (2), strip 20 can be advanced intochannel 12 to a point at which less than 1 cm of strip 20 remainsoutside the entry chosen for insertion, and if, in step (3), adequateelectrophoretic separation is achieved.

If strip 20 is an IPG strip, this latter test an usefully be performedas follows.

Mix 5.0 μL of Serva IEF standard (catalogue no. 39212-01, ServaElectrophoresis GmbH, Heidelberg, Germany) with 120.0 μL of rehydrationbuffer of the following composition: 8.0 M urea, 0.5% ampholytes (3-10IPG buffer, cat. no. 17-6001-11, Amersham Pharmacia Biotech), 2.0% (w/v)CHAPS, 20 mM DTT, 0.0025% (w/v) bromphenol blue. Pipette the solutioninto a channel of the cassette, with the cassette positionedhorizontally. Insert the strip into the channel so that about 3 mmoverextends the channel entries. Occlude the channel entries with covertape and allow the strip to rehydrate for 8 hours. Remove cover tapeand, if present, loading wells. Contact electrodes to anodic andcathodic ends of the strip. Apply a voltage in three steps according tothe following protocol: 250 volts for 15 minutes, ramp from 250-3500volts for 1 hour and 30 minutes, and 3500 volts for 1 hour. Limitcurrent to 1 mA and power to 4 watts in all three steps. Channeldimensions are suitable if discrete marker bands are observable.

Channel entries 14 and 16 will typically, but not invariably, be spacedso that channel 12 engages substantially the entire length of strip 20,as shown e.g. in FIG. 2.

IPG strips are currently available commercially in a variety of lengths.For example, Immobiline DryStrip IPG strips, presently commerciallyavailable from Amersham Pharmacia Biotech, (Piscataway, N.J., USA), areavailable with gel lengths of 70 mm, 110 mm, 130 mm, 180 mm, and 240 mm.ReadyStrip IPG strips, presently commercially available from Bio-Rad(Hercules, Calif., USA), are available with gel lengths of 70 mm, 110and 170 mm.

Thus, in certain presently preferred embodiments of cassette 100 of thepresent invention, channels 12 are fashioned to accommodatesubstantially the entire length of strips with gel lengths of 70 mm, 110mm, 170 mm, 180 mm, and 240 mm in length.

In such commercial IPG strips, the polyester backing typically extendsfor some distance beyond the gel on either end. Thus, channels 12 willtypically have length at least as long as the stated gel length (70,100, 170, 180, or 240 mm), typically with extension of 1 mm, 2 mm, 3 mm,4 mm, 5 mm, or even 6 mm on both ends. Thus, for an IPG strip of nominal70 mm gel length, channel 12 will be at least about 70 mm in length, 72mm in length, 74 mm in length, 76 mm in length, 78 mm in length, andeven 80 or 82 mm in length. In a presently preferred embodiment for IPGstrips of 70 mm stated gel length, channel 12 will be 80 mm in length.

It would be expected that rehydratable strip-based separation media willin the future be available in a variety of lengths, just as they areexpected to be available in a variety of widths and depths, as describedabove. It is, therefore, an aspect of the invention to provide cassettes100 with channels 12 dimensioned to engage prior-cast hydratableelectrophoretic separation media of any chosen length.

As suggested above, significant overextension or underextension ofchannel 12 by strip 20 is undesirable.

For example, if strip 20 extends substantially beyond entry 14, entry16, or both, the overextending portion(s) of strip 20 will be exposed toatmospheric CO₂, obviating an important advantage of the presentinvention. Furthermore, the overextending portion(s) of strip 20 canpermit leakage of ampholyte and/or protein from the strip. Additionally,only that portion of the separation medium lying between the spacedelectrical connections will be functionally available for separation,reducing the functional portion of gel. Finally, the overextendingportion(s) might interfere mechanically with establishment of electricalcommunication properly required for electrophoresis. And when strip 20underextends channel 12, it can prove difficult to establish effectiveelectrical communication with the enclosed strip.

To accommodate these difficulties in a cassette having channels ofnonoptimal length, if strip 20 overextends channel 12, excess can beremoved using scissors or knife; typically, only that portion of strip20 lacking separation media will be so removed. If strip 20 underextendschannel 12, the recessed end can be brought into effective electricalcommunication with the exterior of channel 12 by filling the recessedend with an electrically conductive, channel-filling, material.

Among materials usefully employed to bring the underextended end ofstrip 20 into electrical communication with an entry 14 or 16 ofcassette 100 are materials that can be applied in liquid or semiliquidstate, in which state they can conform in shape to the channel interior,and that thereafter polymerize or gel into a shape-holding phase.

Usefully, the material can be a polymer gel, such as agarose. When soused, the agarose can be rendered molten in the presence ofelectrolyte-containing buffer, such as rehydration solution, applied toentry 14, entry 16, or both as a molten liquid, and thereafter allowedspontaneously to gel with decrease in temperature. Polyacrylamide canalso be used, although in this latter case polymerization of monomersand cross-linkers must be effected by addition of catalyst, as is wellknown in the art.

Usefully, cassette 100 includes a plurality of channels 12. In cases inwhich cassette 100 includes a plurality of channels 12, the current flowaxes of plural channels 12 are usefully substantially parallel to oneanother, and cavities 18 of plural channels 12 are fluidlynoncommunicating with one another except at channel entries 14 and 16.

In such embodiments, channels 12 need not have identical cavity 18dimensions, a single cassette 100 thus accommodating strips 20 ofdifferent dimensions. Typically, however, cavities 18 of plural channels12 will all have the same internal dimensions.

Although cassette 100 is described above as permitting user-directedinsertion of strip 20 into channel 12, it is another aspect of thepresent invention to provide a cassette, as above-described, in whichstrips 20 have already been inserted during manufacture. Such cassettes100 can usefully be disposable.

To facilitate sample application, and in particular to facilitate sampleapplication without cross contamination as among plural channels 12,cassette 100 can usefully include loading wells. FIG. 1 shows oneembodiment of such loading wells.

With reference to FIG. 1, cassette 100 is shown to have two well-formingmembers 22. The two well-forming members define discrete reservoirs,termed loading wells, at each of the six entries 14 and six entries 16,respectively. When cassette 100 is horizontal with well-forming members22 superior to form-retaining member 10, each loading well can maintaina defined maximum volume of fluid in contact with an entry 14 (or entry16) without cross-over fluid contact with adjacent entries.

In cases in which sample to be fractionated is applied after insertionof strip 20, the loading wells permit samples of volume less than themaximum reservoir volume to be applied discretely to individual wells 14(and/or 16) without cross-over contamination. In cases in which sampleis applied in rehydration buffer prior to insertion of strips 20 intochannels 12, the loading wells prevent cross-over contamination bysample displaced from channel 12 during strip insertion.

After sample to be fractionated (such as a protein sample forisoelectric focusing on IPG strips) enters the separation medium ofstrip 20, cross-over contamination among channels 12 is usuallyforeclosed, even if entries 14 are thereafter placed in fluidcommunication with one another and entries 16 are thereafter placed influid communication with one another. Accordingly, well-forming members22 can be removable. Such removal can facilitate subsequent applicationof conductive wicks 24, as shown in FIG. 3A and further described below.

Because well-forming member 22 is typically removed prior toelectrophoresis, there are fewer constraints on the materials from whichit can be constructed than for form-retaining member 10 and, inmultilaminate embodiments of cassette 100, for laminate cover 42.Indeed, well-forming member 22 can be constructed of any material thatis substantially chemically unreactive with the rehydration solution,such as ceramic, quartz, glass, silicon and its derivatives, plastic,natural or synthetic rubber polymers, or mixtures thereof. Amongplastics useful in the construction of well-forming member 22 arepolymethylacrylic, polyethylene, polypropylene, polyacrylate,polymethylmethacrylate, polyvinylchloride, polytetrafluoroethylene,polystyrene, polycarbonate, polyacetal, polysulfone, celluloseacetate,cellulosenitrate, nitrocellulose, polystyrene, polyacrylonitrile,polyurethane, polyamides, polyaniline, and mixtures thereof. Siliconeand its derivatives are also useful.

In certain embodiments, well-forming member 22 can be composed ofelectrically conductive materials; this facilitates “active rehydration”of strip 20. In “active rehydration”, strip 20 is rehydrated in thepresence of a low voltage gradient, approximately 100 V, establishedalong the channel current flow axis of strip 20 between entries 14 and16.

In cases in which active rehydration is desired, well-forming member 22can be composed of an electrically-conductive material, such as anelectrically-conductive polymer, such as a polymer impregnated or dopedwith carbon. After both strip 20 and rehydration solution are applied tochannel 12 (in either order), a cathode is contacted to first conductivewell-forming member 22 and an anode is contacted to second conductivewell-forming member 22 and a voltage applied during the rehydrationperiod. The anode and cathode can be, e.g., an electrode bar, such as isfound on the MultiPhor (Amersham Pharmacia Biotech, Piscataway, N.J.) orBlue Horizon (Serva, Heidelberg, Germany) devices.

When cassette 100 is unitary—that is, having channels 12 formedcompletely within form-retaining member 10—well-forming members 22 canbe attached to form-retaining member 10. When cassette 100 is, instead,multilaminate—e.g., with channels 12 formed in part by a laminate cover42—well-forming members 22 can be attached to laminate cover 42, asshown in FIG. 5.

FIG. 5 is an exploded side perspective view showing well-forming members22 attached adhesively to laminate cover 42 using double-sidedwell-forming member adhesive layer 54. However, as described above withrespect to attachment of laminate cover 42 to form-retaining member 10,which discussion is incorporated herein by reference, well-formingmember 22 can be attached to laminate cover 42 by a variety of bondingmeans well known in the microfabrication arts, including thermalwelding, ultrasonic welding, and application of liquid or partiallycured adhesives, as well as by means of adhesive layers.

Well-forming member 22 can in the alternative be attached to laminatecover 42 by engagement of opposing, matching surfaces, as in a snap, orengagement of tongue with groove, or engagement of tab with slot.

However bonded, well forming members 22 will usefully be reversiblyattached to cassette 100, thus permitting removal of the well-formingmembers prior to electrophoresis. In cases in which attachment is bymeans of a double-sided well-forming member adhesive layer 54, theadhesive layer is usefully designed to adhere more strongly toform-retaining member 10 (or, in multilaminate embodiments, to laminatecover 42) than to well-forming member 22; in such adhesively biasedembodiments, removal of well-forming member 22 will typically leaveadhesive layer 54 on form-retaining member 10 (or laminate cover 42),facilitating application of conductive wicks, as further describedbelow.

Although cross-contamination of samples as among plural channels 12 willtypically be foreclosed by entry of sample into the separation medium ofstrip 20, thus obviating the requirement for continued presence ofwell-forming members 22 during electrophoresis, it can nonetheless beadvantageous further to seal entries 14 and/or 16 after sampleapplication.

In these latter embodiments, sealing is accomplished by application toentries 14 and/or 16 of a material that is electrically conductive, thatcan be applied in a state in which it conforms in shape to the entryand/or loading well, and that thereafter polymerizes or gels into ashape-holding phase. As above, such material can usefully be a polymergel, such as agarose or acrylamide.

In particularly useful approaches, entries 14 and/or 16 are sealed withan amount of material sufficient to fill channel 12 and entry 14 (and/orentry 16) to a level flush with the surface of form-retaining member 10.Such geometry facilitates electrical contact of the anodic and cathodicends of strip 20 directly or indirectly with anode and cathodeelectrodes.

Returning to FIG. 1, cassette 100 can optionally, and usefully, includeribs 40.

Ribs 40 facilitate alignment of laminate cover 42 and well-formingmembers 22 during manufacture of cassette 100. Ribs 40 can alsofacilitate proper operational engagement of cassette 100 by anelectrophoresis chamber or buffer core, as further described below.

Ribs 40 can be machined or molded directly from form-retaining member10, or can be separately constructed and fixed thereto. When separatelyconstructed, ribs 40 are usefully constructed of solid or semisolidmaterials that are readily machined, molded, or etched, and that arechemically compatible—that is, do not suffer substantial degradationupon contact—with electrophoretic buffer systems. Usefully, ribs 40 canbe constructed of materials that are substantially electricallyinsulating, including ceramic, quartz, glass, silicon and itsderivatives, or plastic, or mixtures thereof. Among plastics useful inthe construction of ribs 40 are polymethylacrylic, polyethylene,polypropylene, polyacrylate, polymethylmethacrylate, polyvinylchloride,polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal,polysulfone, celluloseacetate, cellulosenitrate, nitrocellulose,polystyrene, polyacrylonitrile, polyurethane, polyamides, polyaniline,polyester, and mixtures and copolymers thereof.

As noted above, after rehydration of and introduction of sample intostrip 20, strip 20 becomes lodgingly enclosed in cavity 18 of channel12. With strip 20 so enclosed, electrophoresis can then be performed,without removing strip 20 from cassette 100, by applying a voltagegradient to flow current through strip 20 along the channel current flowaxis sufficient to effect electrophoretic separation of analytestherein.

FIG. 3A illustrates one useful, but nonlimiting, approach by whichcassette-enclosed strip 20 is rendered contactable by cathode and anodeelectrodes to complete the necessary electrical circuit.

FIG. 3A is a front perspective view of a cassette of the presentinvention having six channels 12. As shown, a first conductive wick 24contacts strips 20 (present in three of six available channels 12) atentries 14; a second conductive wick 24 contacts strips 20 at entries16.

Wick 24 includes an electrically conductive material. The material neednot be constitutively conductive: it suffices, and indeed typically willbe the case, that wick 24 is conductive when wet. In this latter case,wick 24 can usefully be composed of a bibulous material, such as paper,nitrocellulose, felt, nylon, or derivatives thereof.

As described above, as an alternative or in addition to the presence ofwicks 24, strip 20 can be electrically coupled to cathode and anodeelectrodes through intermediation of electrically conductive polymerssuch as agarose.

As shown in FIG. 3A, first conductive wick 24 can usefully contact eachof plural entries 14, and second conductive wick 24 can usefully contacteach of plural entries 16, facilitating application of current inparallel to plural channels 12. While useful, such geometry is notrequired.

First conductive wick 24 is then contacted with an electrode, serving aseither cathode or anode. The choice as between applying a cathode oranode to wick 24 depends upon the intended electrophoretic technique,the location of sample application, and other conditions well known tothose in the electrophoretic arts. For example, for isoelectric focusingusing IPG strips, where one end of the strip is acidic and the otherbasic, the basi end of the strip is preferably placed in electricalcommunication with the cathodic electrode.

Second conductive wick 24 is then contacted with an electrode (an anodeif first wick 24 is contacted with the cathode, a cathode if first wick24 is contacted with the anode).

Any means of electrode attachment to wicks 24 can be used, as long aseffective electrical communication is established.

In an alternative to use of conductive wicks 24, spaced electricalcommunication with enclosed strip 20 can be effected by direct contactof strip 20 with anode and cathode electrodes. Contact can beaccomplished by passage of anode and cathode electrodes through entries14 or 16, or alternatively by passage of electrodes throughform-retaining member 10 or laminate cover 42 elsewhere than at entries14 and/or 16. As an example of the latter approach, electrodes shaped asblades can be used to pierce laminate cover 42 in embodiments in whichlaminate cover 42 is a flexible film, thereby contacting enclosed strip20 at spaced intervals.

Electrophoresis can thereafter be conducted with cassette 100 in anyphysical orientation. In a particularly useful approach, electrodecontact is effected using an adaptor that permits electrophoresis to beconducted with cassette 100 maintained vertically; as noted above, evenwhen cassette 100 is held vertical, channels 12 of cassette 100 can behorizontal or vertical, as desired.

Returning to FIG. 3A, it is, therefore, another aspect of the inventionto provide an adaptor that permits cassettes 100 of the presentinvention, within which are lodgingly enclosed strips 20, to beelectrophoresed in a vertical direction. It should be noted that evenwhen cassette 100 is itself oriented vertically, channels 12 can stillbe oriented horizontally; in such an orientation, channels 12 ofcassette 100, if present plurally, would be spaced with vertical offsetfrom one another. For clarity, therefore, the term “vertical” isintended to refer to the orientation of the cassette, not the channels.

Electrophoresis of cassette 100 in the vertical dimension has thesignificant advantage of reducing the bench footprint of theelectrophoresis device, freeing up valuable bench space for otherequipment or uses.

Furthermore, modular electrophoresis systems for performing slab gelelectrophoresis in the vertical dimension are well known, see e.g. U.S.Pat. Nos. 5,888,369 and 6,001,233, and are commercially available(Invitrogen, Carlsbad, Calif., USA; Bio-Rad, Hercules, Calif., USA). Inpreferred embodiments, the adaptor of the present invention permitscassettes 100 of the present invention to be electrophoresed in suchexisting modular electrophoresis systems, permitting the efficient useof such prior-purchased equipment for electrophoresis of prior-casthydratable electrophoretic separation media, such as IPG strips.

FIG. 6A is a front perspective view of an adaptor, termed a buffer core,of the present invention (front) operationally aligned with, but not yetcontacting, a cassette of the present invention (rear); operationalcontact is shown in FIG. 6B. As can be seen, buffer core 26 is designedsimultaneously to align cathode electrode wire 31 with cathodic wick 24of cassette 100 and anode electrode wire 32 with anodic wick 24 ofcassette 100.

In the embodiment shown, cathode wire 31 is attached at a first end tocathode contact prong 38; analogously, anode wire 32 is attached at afirst end to anode contact prong 36. Contact prongs 38 and 36 permit theremovable attachment of wires having standard female gender plugs; as iswell known in the electrophoresis arts, the other end of such wires istypically connected to a regulatable power supply.

Also as shown, cathode wire 31 extends from cathode contact prong 38 toserrated support ridge 28 before terminating at a second end, and anodewire 32 extends from anode contact prong 36 to serrated support ridge 30before terminating at a second end. Contact between anode wire, cathodewire, and their respective wicks 24 of the cassettes of the presentinvention is effected, in the embodiment shown, across the serratedsupport ridge, which ridge facilitates tight contact as betweenelectrode wire and conductive wick. In such embodiments, serratedsupport ridges 28 and 30 are typically composed of materials that aresubstantially electrically insulating and substantially inert toelectrophoresis running buffers, for example, of plastic.

Although not shown in either FIG. 6A or FIG. 6B, buffer core 26 can, andtypically will, be operationally aligned and contacted simultaneouslywith a second cassette 100, which in FIG. 6A and FIG. 6B would bepositioned further in front of buffer core 26. So aligned and socontacted, buffer core 26 and cassettes 100 define an internal chamber62, open only at the top, and sealed, except from above, from externalliquids. If the number of strips needed to be electrophoresed can beaccommodated in a single cassette, a “buffer dam”, dimensioned similarlyto cassette 100 but lacking channels 12, can be used to complete buffercore internal chamber 62.

In order to conduct electrophoresis using cassettes and buffer cores ofthe present invention, cassettes 100 (or singular cassette 100 and abuffer dam) are aligned and contacted to buffer core 26. The assembly isthen engaged in electrophoresis buffer chamber 34 which itself, or inconjunction with an additional device, urges cassettes 100 (and/orbuffer dam) into sealable contact with buffer core 26. Such additionalurging device can be a cam-activated clamp, as further described in U.S.Pat. No. 6,001,233, incorporated herein by reference in its entirety.Alternatively, buffer core 26 is first loosely engaged inelectrophoresis buffer chamber 34, and cassettes thereafter aligned,contacted to, and then further urged against buffer core 26.

Fluid-tight contact between buffer core 26 and cassette 100 (and/orbuffer dam) is typically, but optionally, further facilitated by agasket, such as a silicone gasket, fitted into groove 70 of buffer core26.

As noted above, buffer core 26 and cassettes 100 (or singular cassette100 and a buffer dam) in sealed engagement therewith define internalchamber 62. This chamber isolates cathode wire 31 and anode wire 32 fromfluids present external to buffer core 26 in electrophoresis chamber 34(chamber 60), so long as the fluid level in electrophoresis chamber 60does not over op cassettes 100.

Accordingly, electrophoresis chamber 34 can be filled with any chosenliquid solution, to a level that does not overtop cassettes 100, withoutaffecting the electrical circuit. Such fluids can thus usefully serve asa heat sink, reducing the temperature of strips 20 as they are subjectedto current flow in cassettes 100.

Electrophoresis is conducted by attaching, via contact prongs 36 and 38,anode and cathode to a regulatable power supply, and applying a voltagegradient sufficient to flow a current through strip 20, the voltagegradient being sufficient to effect separation of analytes within theseparation medium of strip 20. In cases in which strip 20 is an IPGstrip, proteins, influenced by the voltage gradient, begin to migrateuntil the pI of the protein coincides with the pH on the immobilizedgradient, at which point the focused protein ceases to move.

Upon completion of electrophoresis, strips 20 can be withdrawn fromcavity 18 for further processing. As described earlier, although strips20 can at times be removed upon drying via channel entries 14 and 16,strips 20 will typically be removed by expanding the dimensions ofcavity 18 of channel 12; in multilaminate embodiments of cassette 100,this is accomplished by separating laminate cover 42 from form-retainingmember 10.

The buffer core embodiment above-described is designed to facilitateelectrophoresis of a cassette in which, for each channel presenttherein, channel entries 14 and 16 permit electrical communication withthe channel cavity 18 therebetween through a common surface of cassette100, as is shown, e.g., in FIGS. 1-3.

Such a geometry is not required, however. The invention thus furtherprovides a cassette in which entries 14 and 16 do not open through thesame surface of form-retaining member 10, and a buffer core suited toelectrophoresis of such a cassette. A principal advantage of such ageometry is that it can render the cassette compatible with buffer corespresently sold for slab gel SDS-polyacrylamide gel electrophoresis.

FIG. 7A is a front view, and FIG. 7B a side view, of a cassette 1000 ofthe present invention in which entries 114 and 116 of channels 112respectively open through opposite surfaces of cassette 1000.

Channels 112 of cassette 1000, like channels 12 of cassette 100, are sodimensioned as to movingly engage a prior-cast hydratableelectrophoretic separation medium in its dehydrated state and lodginglyenclose the strip after rehydration.

As should be apparent, in order to conduct electrophoresis usingcassette 1000 as the enclosing member, cathode and anode must establishelectrical communication with strip 20 from opposite sides of cassette1000.

As when entries 14 and 16 open channel 12 to the same face of cassette100, so too electrical communication of channel 112 through entries 114and 116 can be direct, as by through-passage of electrodes throughrespective entries, or indirect, as by intermediation by polymer gelsand/or conductive wicks. Additionally, however, when entries 114 and 116open on opposite sides of cassette 1000, electrical communication can beestablished by contact of anode and cathode electrodes separately to afirst and a second buffer reservoir, which reservoirs in turn separatelycontact entries 114 and 116.

In the latter case, first and second buffer reservoirs must bemaintained in electrical isolation from one another, except by way of acircuit to be completed through the separation medium of strip 20.

Such geometry can readily be effected by sealingly contacting cassettes1000, or a singular cassette 1000 and a buffer dam, to a buffer core126, as further described in commonly-owned U.S. Pat. No. 5,888,369,incorporated herein by reference in its entirety, and as availablecommercially from Invitrogen Corp. (XCell II™ Buffer Core withElectrodes, catalogue no. EI9014X, Invitrogen Corp., Carlsbad, Calif.).

In order to conduct electrophoresis using such system, two cassettes1000 (or a single cassette 1000 and a buffer dam) are lodgingly engagedin operational alignment with buffer core 126, as shown in FIGS. 7C and7D.

The assembly of buffer core and cassettes is then engaged inelectrophoresis buffer chamber 34 which itself, or in conjunction withan additional device, urges cassettes 1000 (and/or buffer dam) intosealable contact with buffer core 126. Such additional device canusefully be a cam-activated clamp, such as that further described inU.S. Pat. No. 6,001,233, incorporated herein by reference in itsentirety. Alternatively, buffer core 126 is first loosely engaged inelectrophoresis buffer chamber 34, and cassettes thereafter aligned,contacted to, and then further urged against buffer core 126.

Fluid-tight contact between buffer core 126 and cassette 1000 (and/orbuffer dam) is typically, but optionally, further facilitated by agasket, such as a silicone gasket, fitted into groove 170 of buffer core126.

Buffer core 126 and cassettes 1000 (or singular cassette 1000 and bufferdam) in sealed engagement therewith define internal chamber 162 which,if cassettes 1000 are not overtopped, is fluidly noncommunicating withelectrophoresis buffer chamber 34. A conductive solution is then addedto internal chamber 162 to a level that (i) contacts cassette entries114 (or 116, as the case may be) that open into chamber 162, and (ii)that does not overtop cassettes 1000. A conductive solution is alsoadded to electrophoresis buffer chamber 34 to a level that (i) contactsthe cassette entries 116 (or 114, as the case may be) that open intochamber 34, and (ii) that does not overtop cassettes 1000.

As further described in commonly-owned U.S. Pat. No. 5,888,369, and wellknown to users of the XCell™ SureLock system, the electrode geometry ofbuffer core 126 effects contact of the anode to internal chamber 126 andcathode to an external reservoir 60 formed in chamber 34, thuspermitting the requisite voltage gradient to be applied across strip 20to effect electrophoresis.

It should be noted that a potential disadvantage of direct contact ofchannels 112 and strips 20 with liquid reservoirs is the increasedlikelihood of ampholyte and/or sample leakage from the separationmedium.

Although the cassettes of the present invention have been particularlydescribed herein above as having at least one prior-formed channel withsufficient dimensional integrity as to permit the lodging by hydrationof prior-cast hydratable separation media engaged there within,prior-formed channels are only one approach to hydratingly lodging suchmedia within an enclosing member.

By way of example only, the enclosing member, if malleable yetshape-retaining, can be wrapped around the strip in its dehydrated form,fashioning a de novo channel which, upon hydration of the strip,lodgingly encloses the rehydrated strip there within.

In a further aspect, the present invention provides kits that facilitatethe practice of the methods of the present invention.

The kits of the present invention comprise at least one enclosing member(cassette) of the present invention, and, as convenient, furthercomprise at least one of prior-cast hydratable electrophoreticseparation media, conductive wicks, containerized buffers—either inliquid form, at use (1×) concentration or higher concentration forfurther dilution, or in dry form to be reconstituted with water ofsuitable quality—buffer cores, and electrophoresis buffer tanks.

EXAMPLE 1 Determination of Channel Tolerances

Three cassettes were manufactured by machining six parallel channelseach into form-retaining plastic slabs, with geometry essentially asshown in FIG. 1. The six channels of the first cassette all were 0.77 mmin depth, with two channels 4.09 mm in width, two channels 0.65 mm inwidth, and two channels 3.35 mm in width. The six channels of the secondcassette all were 0.65 mm in depth, with two channels 4.09 mm in width,two channels 0.65 mm in width, and two channels 3.35 mm in width. Thesix channels of the third cassette all were 0.57 mm in depth, with twochannels 4.09 mm in width, two channels 0.65 mm in width, and twochannels 2.35 mm in width. The channels were rendered fluidly enclosingexcept at terminal entries by application of a flexible laminate coverto each of the three cassettes.

Serva IEF standard, 5 μL, (catalogue no. 39212-01, Serva ElectrophoresisGmbH, Heidelberg, Germany) was mixed with 120 μL of rehydration bufferof the following composition: 8.0 M urea, 0.5% ampholytes (3-10 IPGbuffer, cat. no. 17-6001-11, Amersham Pharmacia Biotech), 2.0% (w/v)CHAPS, 20 mM DTT, 0.0025% (w/v) bromphenol blue. The solution waspipetted into each channel of the three cassettes, with the cassettepositioned horizontally.

An Immobiline DryStrip 3-10 7 cm gel (Amersham Pharmacia Biotech,Piscataway, N.J., USA) was inserted into each channel. The channelentries were occluded with cover tape and the strips allowed torehydrate for 8 hours. Cover tape was removed, as were loading wells ifpresent.

A filter paper wick dampened with water was placed in contact with theextreme ends of the gel portion of the strip at the terminal entries.

Electrodes were contacted to the wick at the anodic and cathodic ends ofthe cassette and a voltage applied in three steps according to thefollowing protocol: 250 volts for 15 minutes, ramp from 250-3500 voltsfor 1 hour and 30 minutes, and 3500 volts for 1 hour. Current waslimited to 1 mA and power to 4 watts in all three steps.

Strips were removed, stained with Coommasie blue stain, aligned, andphotographed.

Results shown in FIG. 8 indicate that even the largest channel, 4.09 mmin width and 0.77 mm in depth, permitted adequate focusing of the ServaIEF standard (left-most lane) in strips with nominal width of 3 mm anddepth of 0.5 mm.

All patents and publications cited in this specification are hereinincorporated by reference as if each had specifically and individuallybeen incorporated by reference herein. Although the foregoing inventionhas been described in some detail by way of illustration and example, itwill be readily apparent to those of ordinary skill in the art, in lightof the teachings herein, that certain changes and modifications may bemade thereto without departing from the spirit or scope of the appendedclaims, which, along with their full range of equivalents, alone definethe scope of invention.

1. A method for performing electrophoresis, comprising: hydratinglylodging a prior-cast hydratable electrophoretic separation medium withinan enclosing member that permits spaced electrical communication withsaid enclosed medium; and then using said spaced electricalcommunication to establish a voltage gradient in said medium sufficientto effect electrophoretic separation of analytes therein.
 2. The methodof claim 1, further comprising the antecedent step of inserting saidprior-cast hydratable electrophoretic separation medium in itsdehydrated state within said enclosing member.
 3. The method of claim 2,wherein said step of hydratingly lodging comprises: contacting saidenclosed dehydrated prior-cast hydratable electrophoretic separationmedium with an aqueous solution for a time sufficient to lodge saidseparation medium within said enclosing member.
 4. The method of claim3, wherein said aqueous solution comprises a sample to be separated insaid prior-cast hydratable separation medium.
 5. The method of claim 1,further comprising a later step of removing said prior-cast hydratableelectrophoretic separation medium from said enclosing member.
 6. Themethod of any of claims 1-5, wherein said prior-cast hydratableelectrophoretic separation medium has an immobilized pH gradient.
 7. Acassette for performing electrophoresis, comprising: means forhydratingly lodging a prior-cast electrophoretic separation mediumwithin an enclosing member; and means for spaced electricalcommunication with said enclosed medium, wherein said spaced electricalcommunication means can be used to establish a voltage gradient in saidenclosed separation medium sufficient to effect electrophoreticseparation of analytes therein.
 8. A cassette for performingelectrophoresis, comprising: a form-retaining member; and at least onechannel, wherein said form-retaining member imparts dimensionalintegrity to said at least one channel; said at least one channel has afirst channel entry, a second channel entry, and a cavity therebetween,said channel cavity being so dimensioned as to permit insertion of ahydratable prior-cast electrophoretic separation medium in itsdehydrated state and lodgingly enclose said hydratable prior-castelectrophoretic separation medium in its rehydrated state; and saidfirst and second channel entries permit spaced electrical communicationwith said channel cavity.
 9. The cassette of claim 8, wherein saidcassette comprises a plurality of said channels.
 10. The cassette ofclaim 8, wherein said form-retaining member contributes the entirecircumferential wall of the cavity of each of said at least one channel.11. The cassette of claim 8, further comprising: a laminate cover,wherein said laminate cover adheres directly or indirectly to saidform-retaining member and contributes at least part of thecircumferential wall of each of said at least one channels.
 12. Thecassette of claim 11, wherein adherence of said laminate cover to saidform-retaining member is reversible.
 13. The cassette of claim 8,further comprising: a first well-forming member, wherein said firstwell-forming member adheres, directly or indirectly, to saidform-retaining member and defines a separate fluid reservoir at thefirst channel entry of each of said at least one channel.
 14. Thecassette of claim 13, further comprising: a second well-forming member,wherein said second well-forming member adheres, directly or indirectly,to said form-retaining member and defines a separate fluid reservoir atthe second channel entry of each of said at least one channel.
 15. Thecassette of claim 14 wherein adherence of said well-forming members tosaid form-retaining member is reversible.
 16. The cassette of claim 8,wherein, for each of said at least one channel, said first and secondchannel entries permit electrical communication with the cavitytherebetween through a common surface of said cassette.
 17. The cassetteof claim 8, wherein, for each of said at least one channel, said firstand second channel entries permit electrical communication with thecavity therebetween through separate surfaces of said cassette.
 18. Thecassette of any of claims 8-17, further comprising: at least oneprior-cast hydratable electrophoretic separation medium, wherein each ofsaid at least one prior-cast separation medium is engaged in a separatechannel of said cassette.
 19. A kit for electrophoresing prior-casthydratable electrophoretic separation media, comprising: a cassetteaccording to claim 9; and at least one prior-cast hydratableelectrophoretic separation medium suitably dimensioned as to behydratingly lodgeable in said cassette.
 20. A kit for electrophoresingprior-cast hydratable electrophoretic separation media, comprising: acassette according to claim 9; and at least one conductive wick.
 21. Abuffer core for vertical electrophoresis of prior-cast hydratableseparation media, comprising: a substantially inflexible frame, saidframe having a first cassette engagement face; an anode; and a cathode,wherein said anode and cathode are positioned to effect simultaneousspaced contact with a common surface of a cassette operationallycontacted to said first cassette engagement face.
 22. A buffer coreaccording to claim 21, further comprising: a second cassette engagementface; wherein operational engagement of a first and second cassetterespectively to said first and second frame engagement faces creates achamber internal to said frame that is sealed on 5 sides; wherein saidcathode and said anode are each in electrical communication with theinterior of said internal chamber; and wherein operational contact of afirst and second cassette to said respective first and second frameengagement faces causes spaced contact of said anode and said cathode toa common surface of at least one of said cassettes.